The long-term goal of this research is to develop and apply purpose-inspired chemical tools for detecting, quantifying, and delivering biological hydrogen sulfide (H2S). Hydrogen sulfide is now accepted as an important physiological mediator and signaling agent, joining NO and CO as an endogenous gasotransmitter. H2S plays important roles in various conditions associated with human health including diabetes, hypertension, atherosclerosis, inflammation, neurodegeneration, sepsis, and asthma. Upon enzymatic production, H2S exerts its action on different molecular targets, including ion channels and signaling proteins through reaction with protein thiols and transition-metal centers. Despite the diverse and important biochemical roles of H2S, limited methods are available for detecting, quantifying, or delivering biologically-relevant H2S concentrations. Although many of the reported chemical tools for H2S work well in non-biological settings, few of these tools have proven sufficiently sharp to make the transition from test tubes to real biological contexts. To address these unmet needs, the objectives in this proposal are to develop and refine purpose-inspired chemical tools for biological H2S detection, quantification, and delivery and to apply these tools to investigate host-microbial interactions in live zebrafish. The rational for this wor is that successful completion of the proposed Aims will provide robust chemical tools and provide a positive impact toward studying and understanding the multifaceted roles of H2S in biology. Motivated by proof-of-concept preliminary results supporting the research design, specific aims include: 1) Development and application of selective reporters for H2S; 2) Development and application of traceable, slow-release H2S donors; 3) Functional imaging of H2S and microbial colonization dynamics in zebrafish. Objectives outlined in the first Aim include new platforms to translate H2S imaging into biologically-relevant concentration ranges by using bright pH insensitive chromophores, targeted H2S sensors, and signal amplification methods to lower the detection limits of H2S probes. Objectives outlined in the second Aim include new innovative strategies for coupling H2S delivery with an optical readout to facilitate non- invasive, real-time monitoring of H2S delivery to bridge the gap between cuvette- and context-based measurements of H2S release. Objectives outlined in the third Aim include imaging H2S production in conventional, germ-free, and monobacterial zebrafish, and determining the role of H2S on microbial gut colonization dynamics. The proposed research is significant because the outlined approaches directly address current unmet needs and limitations in the field related to H2S imaging, quantification, and delivery. Successful completion of the proposed Aims will provide a positive impact in the field of H2S biochemistry and will result a greater understanding of the important and multifaceted roles of H2S associated with human health.